ECAOR DEBATE

-3 -3

pletely closed at 10 mol m . These results are in broad agreement with those reported previously on Commelina communis epidermal strips, by Snaith & Mansfield (1982b) and Wilson (1981). To look for possible interactions between calcium and abscisic acid, epidermal pieces were incubated in the presence of low concentrations of calcium (0.1 and

___ ^ ""6 5______ _3

0.25 mol m ) and ABA (10 and 10 mol m ), and the results are shown in Fig. 3.2. In this experiment there was a very small reduc-

"6 "“3

tion in aperture at 10 mol m ABA; experience has shown that there is a slight variation in the sensitivity of stomata to ABA in different experiments (compare histogram B of Fig. 3.2 with Fig. 3.1 in which no

~3

effect of 10 mol m ABA was observed. A possible explanation for such variations is given in the previous Chapter). The little effect observed at 10 6 mol m 3 ABA was amplified when it was applied in the

- 3

presence of 0.1 mol m calcium and it became significant. This clearly shows a synergistic response of stomata to ABA and calcium, and the examination of data for other concentrations, with the exception of 10-5 mol m ~3 ABA + 0 . 2 5 mol m 3 Ca2+, supports this conclusion.

That exception rather shows an antagonism between the two agents.

This is attributed to some of the stomata being closed and thus unable to respond further. A similar situation was observed in the next experiment in which the interaction was examined further in a 3 x 5 factorial design. The analysis of variance revealed a highly sig­ nificant (P <0.001) interaction between ABA and calcium, and the appearance of the isometric projection of the data (Fig. 3.3) suggests

61. 20 16 12 8 4 0 Figure 3.3

Isometric projection of the results of a 3 x 5 factorial experiment showing effects of ABA and calcium in preventing opening of stomata on isolated epidermis. Means of 90 measurements of individual stomata.

62.

-6 -4 -3

that this occurs mainly in the range of 10 to 10 mol m ABA. The strong suppression of opening caused by ABA and calcium when present simultaneously, was closely associated with a reduction in guard cell K content. Therefore, the observed greater-than-additive effects may be explained in terms of their effects on ion accumulation. Since the net accumulation is determined by the degree of both influx and efflux, an inhibition of influx by one agent occurring simultaneously with a stimulation of efflux by the other could be manifested as a synergistic suppression of guard cell turgor, because the relationship between turgor and the influx/efflux process may not be linear (MacRobbie & Lettau, 1980b). The synergism observed between ABA and the synthetic auxin, naphth-l-ylactic acid, has tentatively been explained in this way (Snaith & Mansfield, 1984).

Alternatively, if the action of one agent depended in some way on the presence of the other, that could lead to synergism. For example, if the attachment of ABA to its recently discovered binding protein

(Hornberg & Weiler, 1984), resulted in an increase in membrane perme­ ability to calcium, and the subsequent increase in intracellular calcium affected the guard cell K channels reported by Schroeder, Hedrich & Fernandez (1984), then a synergistic effect of ABA and calcium would be expected. This seems to be a feasible explanation for this syner­ gism and, therefore, it suggests that the action of ABA is dependent on the availability of calcium ions in the free space. Perhaps, one might then argue that ABA would not exert its effects on the guard cells with no added calcium in the incubation medium. However, as it has already been pointed out, there is likely to be free calcium in the apoplast in sufficient amounts and that may account for the observed effects of ABA when calcium was not provided in the external medium.

63. E 3. <D i— u. <D Cl a O o E o 00

Control 0*1 mol m~3 IO"5 mol m-3 0*1 mol m-3 colcium AB A calcium

4 IO” 5mol rrT3 ABA

EGTA: - + — + — + “ +

Figure 3.4

Effect of 2 mol m”3 EGTA on the stomatal responses to ABA and/or Ca C ^ . Epidermal pieces were incubated for 3 h in the absence of C02/ in light, at 25 ± 1°C. The treatments were: control,

0.1 mol m-3 CaCl„, 10 mol m" ABA and 0.1 mol m CaCl + 10 2

__ -3

mol m ABA. Each treatment was given with or without 2 mol m EGTA. Means of 90 measurements of individual stomata with 95% confidence limits.

action of the removal of apoplastic calcium using the chelating agent _3

EGTA. Data m Figure 3.4 show that when 2 mol m EGTA was present in the incubation medium, the individual and synergistic suppression

2+

of stomatal opening by ABA and Ca was abolished, and the opening in each case was greater than that in the control without EGTA. The stimulation of opening in the controls by EGTA was probably due to the removal of endogenous apoplastic calcium which might have

prevented opening to some degree.

In view of the results presented in this Chapter, it appears that free calcium in the apoplast is essential to enable guard cells to respond to ABA. Although the molecular basis of the synergism

2+

is not known, ABA seems to increase cytosolic Ca concentration. The subsequent action of calcium ions in the guard cells is as yet unknown, but it can be speculated that calmodulin may be involved, and this possibility will be examined in the following Chapter.